African trypanosomiasis has two major manifestations: the human disease known as Sleeping Sickness and the animal disease Nagana (meaning loss of spirits in the Zulu language). Animal trypanosomiasis is endemic in equatorial Africa, where it is transmitted, among humans and animals, by Glossina, the Tsetse. The native African fauna are almost universally infected, providing a vast reservoir of potential human pathogens. Focal human epidemics are a serious reality and an increasing threat. If untreated, trypanosomiasis is rapidly fatal and is currently estimated to kill around 100,000 people annually. The available treatments are unsatisfactory and often ineffective. One of the major reasons for the persistence of African trypanosomes is their unique and deadly efficient mechanism to evade the mammalian immune response. Each trypanosome shields its surface membrane with a replaceable molecular 'coat' composed of a single species of Variant Surface Glycoprotein (VSG). In a process known as Antigenic Variation, the majority of the trypanosome population is destroyed by the immune responses to the VSG coat, but individual trypanosomes switch their VSG, using a repertoire of hundreds of VSG genes, evade destruction, and seed successive waves of parasitemia. By developing methods for culturing and genetically manipulating trypanosomes in vitro, we have moved the study of antigenic variation from observation to experimentation. To be expressed, a VSG gene has to be located in a polycistronic telomeric Expression Site (ES), but this is not sufficient for transcription. There are more than 20 potential ESs, but only one is active at any time. The focus of this proposal is to identify mechanisms that regulate ES transcription. We will continue to characterize proteins that interact with the ES promoter. The most persuasive current model for the exclusiveness of ES transcription is that, to be productively transcribed, the ES has to be at a specific sub-nuclear location, dubbed the ES Body. We will identify factors that influence the nuclear localization of silent and active ESs, focusing on the potential role of the 50-bp repeat domains that are only found upstream of ES promoters. We will also explore the possibility of developing 'forward' genetic approaches that will be essential for identifying novel genes that are undoubtedly involved in ES silencing and switching.